Selection of potential Cytotoxic T lymphocyte epitopes conserved among related Orthopoxviruses using a Bioinformatic Appoach

Poster number: 9

Sheila Tuyet Tang (1), Morten Nielsen (1), Mette Volby Larsen (1), Kasper Lambert2, Søren Buus (2), Mogens Claesson (2), Ole Lund (1)

  1. Center for Biological Sequence Analysis, Technical University of Denmark, BioCentrum-DTU, Building 208, 2800, Lyngby, Denmark
  2. Division of Experimental Immunology, Institute of Medical Microbiology and Immunology (IMMI), University of Copenhagen, Building 18.3, Panum Institute, Blegdamsvej 3, 2200 Copenhagen N, Denmark.

CD8+ T lymphocytes have been shown to be involved in controlling poxvirus infection, but until recently only very few protective cytotoxic T-lymphocyte (CTL) epitopes are defined/predicted for Variola virus, the causative agent of smallpox, or other related orthopoxviruses. In this study Artificial neural networks (ANNs) were used to predict CTL epitopes from publicly available full-length protein sequences of Variola virus. Epitopes were selected based on their novelty, conservation and being immunomodulators.

The top 15 CTL epitopes for each of 14 different HLA alleles were predicted and synthesized synthetically and their binding affinity to the relevant HLA molecules will be tested experimentally by in vitro binding assay to HLA-A and HLA-B molecues.

We will proceed with CTL assay to examine if cytotoxic T cells from HLA typed blood donors can react to the selected peptides. The donors will be tested for response against up to 15 peptides for each of their maximally 2 different HLA-A alleles and 2 different HLA-B alleles.

The bioinformatics approach of predicting potential CTL epitopes candidates is relatively cheap to perform and will lower the amount of costly laboratory experiments and thus only focused on a little subset of possible CTL epitopes.

Identification of T cell epitopes on Variola virus will enable further studies to increase our understanding of the role of CD8+ T cells in pox virus infection and assist in the design of new protective strategies like a DNA polytope vaccine design.


Seven orthopoxvirus strains were selected to be included in this study: Variola virus India (VARC-IND), modified Vaccinia virus (VACV)-Ankara; VACV-Western Reserve, VACV Copenhagen and finally two cowpox strains were selected; Brighton Red and strain GRI-90.

A program “genediff” was used to find conserved orthologues proteins shared between the different selected orthopoxvirus strains. This generated a list of 157 orthologues proteins. These orthologues proteins were subdued to the following selection criteria to find pox CTL epitopes within immunomodulatory proteins (22) and among all conserved proteins:

1. Selection of epitopes in immunomodulator proteins

The whole genome of VARV-IND was scanned in order to find viral proteins which interfere with the host immune system and thus affect the pathogenesis.

Twenty-two of these immunomodulators were found to be conserved in the list of orthologues proteins for all the selected strains of orthopoxviruses. ANN prediction method was used for predicting pox CTL epitope within these immunnomodulators. Pox CTL epitopes were predicted for the following HLA-A and HLA-B supertypes: A1, A2, A24, A26, A3, B27, B39, B40, B58, B62, B7, respectively. The list of supertypes is representing the different HLA alleles in the world.

2. Selection of conserved epitopes

Concerved predicted CTL epitopes were selected among the list of homologues genes within the seven selected strains of orthopoxviruses.

NetCTL ( was used to predict CTL epitopes. This method combines predictions of proteosomal cleavage, TAP transport and MHC binding.